The protective mechanisms of mammalian cells against exogeneous and endogenous stressors that generate harmful free radicals employ the antioxidant co-enzyme, glutathione (GSH). GSH is important in maintaining the structural integrity of cell and organelle membranes and in the synthesis of microtubules and macromolecules. See C. D. Klassen et al. Fundamental and Applied Toxicology, 5, 806 (1985). Stimulation of GSH synthesis in rat renal epithelial cells and stomach cells has been found to protect the cells from the toxic effects of cyclophosphamide and serotonin, respectively. Conversely, inhibition of glutathione synthesis and glutathione depletion has been found to have the following effects: (a) decreased cell viability, (b) increased sensitivity of cells to the effects or irradiation, (c) increased sensitivity of tumor cells to peroxide cytolysis, (d) decreased synthesis of prostaglandin E and leukotriene C and (e) selective destruction of trypanosomes in mice.
Biosynthesis of glutathione (GSH) involves two sequential reactions that utilize ATP and that are catalyzed by the enzymes γ-glutamylcysteine synthetase and glutathione synthetase (GSH-synthetase) using the three precursor amino acids L-glutamic acid, L-cysteine, and glycine, as shown in FIG. 1.
All substrate-level reactants occur at near enzyme-saturating concentrations in vivo with the exception of L-cysteine, whose cellular concentration is exceedingly low. Therefore, the first reaction in which L-cysteine is required, i.e., the synthesis of γ-L-glutamyl-L-cysteine, is the rate-limiting step of glutathione biosynthesis. Thus, the availability of intracellular L-cysteine is a critical factor in the overall biosynthesis of GSH, are sufficient stores of ATP.
In the synthesis of ATP via the nucleotide salvage pathway, the nucleotide precursors that may be present in the tissue are converted to AMP and further phosphorylated to ATP. Adenosine is directly phosphorylated to AMP, while xanthine and inosine are first ribosylated by 5-phosphoribosyl-1-pyrophosphate (PRPP) and then converted to AMP.
Ribose is found in the normal diet only in very low amounts, and is synthesized within the body by the pentose phosphate pathway. In the de novo synthetic pathway, ribose is phosphorylated to PRPP, and condensed with adenine to form the intermediate adenosine monophosphate (AMP). AMP is further phosphorylated via high energy bonds to form adenosine diphosphate (ADP) and ATP.
During energy consumption, ATP loses one high energy bond to form ADP, which can be hydrolyzed to AMP. AMP and its metabolites adenine, inosine and hypoxanthine are freely diffusible from the muscle cell and may not be available for resynthesis to ATP via the salvage pathway.
The availability of PRPP appears to control the activity of both the salvage and de novo pathways, as well as the direct conversion of adenine to ATP. Production of PRPP from glucose via the pentose phosphate pathway appears to be limited by the enzyme glucose-6-phosphate dehydrogenase (G6PDH). Glucose is converted by enzymes such as G6PDH to ribose-5-phosphate and further phosphorylated to PRPP, which augments the de novo and salvage pathways, as well as the utilization of adenine.
Many conditions produce hypoxia. Such conditions include acute or chronic ischemia when blood flow to the tissue is reduced due to coronary artery disease or peripheral vascular disease where the artery is partially blocked by atherosclerotic plaques. In U.S. Pat. No. 4,719,201, it is disclosed that when ATP is hydrolyzed to AMP in cardiac muscle during ischemia, the AMP is further metabolized to adenosine, inosine and hypoxanthine, which are lost from the cell upon reperfusion. In the absence of AMP, rephosphorylation to ADP and ATP cannot take place. Since the precursors were washed from the cell, the nucleotide salvage pathway is not available to replenish ATP levels. It is disclosed that when ribose is administered via intravenous perfusion into a heart recovering from ischemia, recovery of ATP levels is enhanced.
Transient hypoxia frequency occurs in individuals undergoing anesthesia and/or surgical procedures in which blood flow to a tissue is temporarily interrupted. Peripheral vascular disease can be mimicked in intermittent claudication where temporary arterial spasm causes similar symptoms. Finally, persons undergoing intense physical exercise or encountering high altitudes may become hypoxic. U.S. Pat. No. 6,218,366 discloses that tolerance to hypoxia can be increased by the administration of ribose prior to the hypoxic event.
Hypoxia or ischemia can also deplete GSH. For example, strenuous aerobic exercise can also deplete antioxidants from the skeletal muscles, and sometimes also from the other organs. Exercise increases the body's oxidative burden by calling on the tissues to generate more energy. Making more ATP requires using more oxygen, and this in turn results in greater production of oxygen free radicals. Studies in humans and animals indicate GSH is depleted by exercise, and that for the habitual exerciser supplementation with GSH precursors may be effective in maintaining performance levels. See L. L. Ji, Free Rad. Biol. Med., 18, 1079 (1995).
Tissue injury, as from burns, ischemia and reperfusion, surgery, septic shock, or trauma can also deplete tissue GSH. See, e.g., K. Yagi, Lipid Peroxides in Biology and Medicine, Academic Press, N.Y. (1982) at pages 223-242; A. Blaustein et al., Circulation, 80, 1449 (1989); H. B. Demopoulos, Pathology of Oxygen, A. P. Autor, ed., Academic Press, N.Y. (1982) at pages 127-128; J. Vina et al., Brit. J. Nutr., 68, 421 (1992); C. D. Spies et al., Crit. Care Med., 22, 1738 (1994); B. M. Lomaestro et al., Annals. Pharmacother., 29, 1263 (1995) and P. M. Kidd, Alt. Med. Res., 2, 155 (1992).
It has been hypothesized that delivery of L-cysteine to mammalian cells can elevate GSH levels by supplying this biochemical GSH precursor to the cell. However, cysteine itself is neurotoxic when administered to mammals, and is rapidly degraded. In previous studies, it was shown that N-acetyl-L-cysteine, L-2-oxothiazolidine-4-carboxylate, as well as 2(R,S)-n-propyl-, 2(R,S)-n-pentyl and 2(R,S)-methyl-thiazolidine-4R-carboxylate can protect mice from heptatotoxic dosages of acetaminophen. See H. T. Nagasawa et al., J. Med. Chem., 27, 591 (1984) and A. Meister et al., U.S. Pat. No. 4,335,210. L-2-Oxothiazolidine-4-carboxylate is converted to L-cysteine via the enzyme 5-oxo-L-prolinase. As depicted in FIG. 2, compounds of formula 1, e.g., wherein R═CH3, function as prodrug forms of L-cysteine (2), liberating this sulfhydryl amino aciuc by nonenzymatic ring opening and hydrolysis. However, the dissociation to yield L-cysteine necessarily releases an equimolar amount of the aldehyde (3), RCHO. In prodrugs in which R is an aromatic or an alkyl residue, the potential for toxic effects is present.
U.S. Pat. No. 4,868,114 discloses a method comprising stimulating the biosynthesis of glutathione in mammalian cells by contacting the cells with an effective amount of a compound of the formula (1):
wherein R is a (CHOH)nCH2OH and wherein n is 1-5. The compound wherein n is 3 is 2(R,S)-D-ribo-(1′,2′,3′,4′-tetrahydroxybutyl)thiazolidone-4(R)-carboxylic acid (Ribose-Cysteine, RibCys). Following in vivo administration, RibCys releases cysteine by non-enzymatic hydrolysis. RibCys has been demonstrated to be effective to protect against acetaminophen-induced hepatic and renal toxicity. A. M. Lucus, Toxicol. Pathol., 28, 697 (2000). RibCys can also protect the large and small bowel against radiation injury. See M. P. Caroll et al., Dis. Colon Rectum, 38, 716 (1995). These protective effects are believed to be due to the stimulation of GSH biosynthesis, which elevates intracellular GSH. However, a need exists for methods to restore or maintain intracellular GSH stores in mammalian tissues subjected to hypoxic conditions in which the ATP stores necessary to drive the biosynthesis of GSH and its precursors are depleted.